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J V Small

Publications and source records attributed to J V Small.

At least 37 records · Page 2Linked to original sources
PubMed1996

Actin and the coordination of protrusion, attachment and retraction in cell crawling.

To crawl over a substrate a cell must first protrude in front, establish new attachments to the substrate and then retract its rear. Protrusion and retraction utilise different subcompartments of the actin cytoskeleton and operate by different mechanisms, one involving actin polymerization and the other myosin-based contraction. Using as examples the rapidly locomoting keratocyte and the slowly moving fibroblast we illustrate how over expression of one or the other actin subcompartments leads to the observed differences in motility. We also propose, that despite these differences there is a common coordination mechanism underlying the genesis of the actin cytoskeleton that involves the nucleation of actin filaments at the protruding cell front, in the lamellipodium, and the relocation of these filaments, via polymerization and flow, to the more posterior actin filament compartments.

Actins↗
PubMed1996

Coordination of protrusion and translocation of the keratocyte involves rolling of the cell body.

We have investigated the relationship between lamellipodium protrusion and forward translocation of the cell body in the rapidly moving keratocyte. It is first shown that the trailing, ellipsoidal cell body rotates during translocation. This was indicated by the rotation of the nucleus and the movement of cytoplasmic organelles, as well as of exogenously added beads used as markers. Activated or Con A-coated fluorescent beads that were overrun by cells were commonly endocytosed and rotated with the internal organelles. Alternatively, beads applied to the rear of the cell body via a micropipette adhered to the dorsal cell surface and also moved forward, indicating that both exterior and underlying cortical elements participated in rotation. Manipulation of keratocytes with microneedles demonstrated that pushing or restraining the cell body in the direction of locomotion, and squeezing it against the substrate, which temporarily increased the intracellular pressure, did not effect the rate of lamellipodium protrusion. Rotation and translocation of the cell body continued momentarily after arrest of lamellipodium protrusion by cytochalasin B, indicating that these processes were not directly dependent on actin polymerization. The cell body was commonly flanked by phase-dense "axles," extending from the cell body into the lamellipodium. Phalloidin staining showed these to be comprised of actin bundles that splayed forward into the flanks of the lamellipodium. Disruption of the bundles on one side of the nucleus by traumatic microinjection resulted in rapid retraction of the cell body in the opposite direction, indicating that the cell body was under lateral contractile stress. Myosin II, which colocalizes with the actin bundles, presumably provides the basis of tension generation across and traction of the cell body. We propose that the basis of coupling between lamellipodium protrusion and translocation of the cell body is a flow of actin filaments from the front, where they are nucleated and engage in protrusion, to the rear, where they collaborate with myosin in contraction. Myosin-dependent force is presumably transmitted from the ends of the cell body into the flanks of the lamellipodium via the actin bundles. This force induces the spindle-shaped cell body to roll between the axles that are created continuously from filaments supplied by the advancing lamellipodium.

Animals↗
PubMed1996

The SM 22 promoter directs tissue-specific expression in arterial but not in venous or visceral smooth muscle cells in transgenic mice.

The transcriptional signals underlying smooth muscle differentiation are currently unknown. We report here the complete sequence and characterization of the single mouse gene for the smooth muscle-specific protein SM 22 and the transcriptional activity of its promoter in cultured smooth muscle cells in vitro and in transgenic mice. In the transgenic animals, promoter constructs ranging in length from 445 to 2126 bp directed reporter expression initially in the heart and the somites of embryos and subsequently in the arteries of the vascular system, but in none of the visceral muscles, nor in the veins. Expression in the heart was spatially restricted to the presumptive right ventricle and outflow tract and disappeared in the adult. Likewise, expression in the somites was only transitory and was not observed after about 14.5 days post coitum in the embryo. In the adult mouse, SM 22 promoter activity persisted in the smooth muscle cells of the arteries and was still notably absent from other smooth muscles, despite the ubiquitous presence of the endogenous SM 22 protein. These findings on the transcriptional activity of a smooth muscle promoter in vivo reveal the existence of different differentiation programmes for smooth muscle cells in the veins and the arteries and raise the expectation of a further subdivision of programmes among the visceral muscles.

Animals↗
PubMed1995

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres.

Calponin (4.1-5.9 microM, pig stomach) inhibited maximal shortening velocity (Vmax) by 20-25% with only minor influence on force in skinned smooth muscle from guinea-pig taenia coli activated at different Ca2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the N-terminal 1-228 amino acids engineered using a mouse cDNA construct (5.4 microM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin-myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross-bridge time spent in the attached state.

Actins↗
PubMed1995

Structure-function relationships in smooth muscle: the missing links.

Smooth muscle cells have developed a contractile machinery that allows them to exert tension on the surrounding extracellular matrix over their entire length. This has been achieved by coupling obliquely organized contractile filaments to a more-or-less longitudinal framework of cytoskeletal elements. Earlier structural data suggested that the cytoskeleton was composed primarily of intermediate filaments and played only a passive role. More recent findings highlight the segregation of actin isotypes and of actin-associated proteins between the contractile and cytoskeletal domains and raise the possibility that the cytoskeleton performs a more active function. Current efforts focus on defining the relative contributions of myosin cross-bridge cycling and actin-associated protein interactions to the maintenance of tension in smooth muscle tissue.

Cytoskeleton↗
PubMed1995

Getting the actin filaments straight: nucleation-release or treadmilling?

The dynamic turnover of actin filaments plays a central role in the locomotion of metazoan cells. Based on results obtained with actin labelled with a caged fluorescent probe, Theriot and Mitchison proposed a 'nucleation-release' model for the fast-moving fish keratocyte, which predicts the existence of short non-oriented filaments in the motile lamellipodium. More recent structural data on keratocyte cytoskeletons do not support this model, but are consistent with the treadmilling of long actin filaments of graded length. Taken together with Theriot and Mitchison's demonstration that the cytoskeleton remains stationary relative to the substrate in the moving keratocyte, the structural data raise the possibility that a lateral flow of filaments plays a role in lamellipodia motility.

Journal Article↗
PubMed1995

Actin filament organization in the fish keratocyte lamellipodium.

From recent studies of locomoting fish keratocytes it was proposed that the dynamic turnover of actin filaments takes place by a nucleation-release mechanism, which predicts the existence of short (less than 0.5 microns) filaments throughout the lamellipodium (Theriot, J. A., and T. J. Mitchison. 1991. Nature (Lond.). 352:126-131). We have tested this model by investigating the structure of whole mount keratocyte cytoskeletons in the electron microscope and phalloidin-labeled cells, after various fixations, in the light microscope. Micrographs of negatively stained keratocyte cytoskeletons produced by Triton extraction showed that the actin filaments of the lamellipodium are organized to a first approximation in a two-dimensional orthogonal network with the filaments subtending an angle of around 45 degrees to the cell front. Actin filament fringes grown onto the front edge of keratocyte cytoskeletons by the addition of exogenous actin showed a uniform polarity when decorated with myosin subfragment-1, consistent with the fast growing ends of the actin filaments abutting the anterior edge. A steady drop in filament density was observed from the mid-region of the lamellipodium to the perinuclear zone and in images of the more posterior regions of lower filament density many of the actin filaments could be seen to be at least several microns in length. Quantitative analysis of the intensity distribution of fluorescent phalloidin staining across the lamellipodium revealed that the gradient of filament density as well as the absolute content of F-actin was dependent on the fixation method. In cells first fixed and then extracted with Triton, a steep gradient of phalloidin staining was observed from the front to the rear of the lamellipodium. With the protocol required to obtain the electron microscope images, namely Triton extraction followed by fixation, phalloidin staining was, significantly and preferentially reduced in the anterior part of the lamellipodium. This resulted in a lower gradient of filament density, consistent with that seen in the electron microscope, and indicated a loss of around 45% of the filamentous actin during Triton extraction. We conclude, first that the filament organization and length distribution does not support a nucleation release model, but is more consistent with a treadmilling-type mechanism of locomotion featuring actin filaments of graded length. Second, we suggest that two layers of filaments make up the lamellipodium; a lower, stabilized layer associated with the ventral membrane and an upper layer associated with the dorsal membrane that is composed of filaments of a shorter range of lengths than the lower layer and which is mainly lost in Triton.

Actins↗
PubMed1994

Beta-actin specific monoclonal antibody.

Using a synthetic peptide mimicking the NH2-terminus of beta-actin we have raised a monoclonal antibody specific for this cytoplasmic actin isoform. Specificity of the antibody was demonstrated by its labelling of the actin polypeptide only in tissues containing the beta isoform, by its exclusive recognition of the synthetic beta-actin peptide amongst those mimicking all six vertebrate isoactins, and by its selective recognition of the beta-actin spot in two-dimensional electrophoresis gels of smooth muscle extracts. The antibody bound to actin filaments in both living and fixed fibroblasts where it labelled the stress fiber bundles and, more predominantly, the peripheral actin rich lamellipodia. The characteristics of the antibody indicate that it should serve as a useful tool for studying isoactin distribution and function.

Actins↗
PubMed1994

Lamellipodia architecture: actin filament turnover and the lateral flow of actin filaments during motility.

Consideration of the arrangement of actin filaments in the lamellipodia of crawling cells indicates that, in addition to a rearward flow of the actin cytoskeleton due to treadmilling, there is a lateral flow of filaments in both directions. The existence of such a lateral flow of actin filaments is supported by observation of the lateral movement of actin filament bundles in fibroblasts and by fluorescence photoactivation data. In addition to explaining the formation, dispersal and lateral movement of filament bundles, lateral filament flow could add a further velocity component to the centripetal flow of cytoplasm. As such, lateral filament flow may explain the discrepancies in the rates of the rearward flow of actin, cytoplasmic materials and dorsal particles.

Actins↗
PubMed1994

Calponin is localised in both the contractile apparatus and the cytoskeleton of smooth muscle cells.

Calponin and caldesmon are two thin filament-binding proteins found in smooth muscle that have both been attributed a role in modulating the interaction of actin and myosin. Using high-resolution dual-label immunocytochemistry we have determined the distribution of calponin relative to the contractile and cytoskeletal compartments of the smooth muscle cell. We show, using chicken gizzard smooth muscle, that calponin occurs in the cytoskeleton, with beta-cytoplasmic actin, filamin and desmin, as well as in the contractile apparatus, with myosin and caldesmon. According to the observed labelling intensities, calponin was more concentrated in the cytoskeleton and it was additionally localised in the cytoplasmic dense bodies as well as in the adhesion plaques at the cell surface, which both harbour the beta-cytoplasmic isoform of actin. It is probable that these results explain earlier conflicting reports on the composition of smooth muscle thin filaments and suggest that calponin, together with a Ca(2+)-receptor protein, could just as likely serve a role in the cytoskeleton of smooth muscle as in the contractile apparatus.

Animals↗
PubMed1994

Actin isoform compartments in chicken gizzard smooth muscle cells.

Differentiated smooth muscle cells typically contain a mixture of muscle (alpha and gamma) and cytoplasmic (beta and gamma) actin isoforms. Of the cytoplasmic actins the beta-isoform is the more dominant, making up from 10% to 30% of the total actin complement. Employing an antibody raised against the N-terminal peptide specific to beta-actin, which labels only the beta-isoform on two-dimensional gel immunoblots, we have shown that this isoform has a restricted localisation in smooth muscle. Using double-label immunofluorescence and immunoelectron microscopy of ultrathin sections of chicken gizzard, beta-actin was localised in the dense bodies and in longitudinal channels linking consecutive dense bodies that were also occupied by desmin. It was additionally found in the membrane-associated dense plaques, but was excluded from the actomyosin-containing regions of the contractile apparatus. Taken together with earlier results these findings identify a cytoskeletal compartment containing intermediate filaments, cytoplasmic actin and the actin cross-linking protein filamin. Using an antibody specific only for muscle actin, labelling was found generally around the myosin filaments of the contractile apparatus, but was absent from the core of the dense bodies that contained beta-actin. Thus, if dense bodies act as dual-purpose anchorage sites, for the cytoskeletal actin and the contractile actin, the thin filaments of the contractile apparatus must be anchored at the periphery of the dense bodies. A model of the structural organisation of the cell is presented and the possible roles of the cytoskeleton are discussed.

Actins↗
PubMed1993

Mammalian calponin. Identification and expression of genetic variants.

Calponin is a smooth muscle specific, actin-, tropomyosin- and calmodulin-binding protein thought to be involved in some way in the regulation or modulation of contraction. Here we describe the cloning and bacterial expression of two calponin species from murine and porcine smooth muscle tissues. Primary and secondary structural analyses of the deduced amino acid sequences revealed a high degree of homology to avian calponin with the exception of a short and variable C-terminal segment. The sequence data demonstrate that the two mammalian calponin variants do not arise via alternative splicing but are encoded by different genes.

Amino Acid Sequence↗
PubMed1993

Variable and constant regions in the C-terminus of vinculin and metavinculin. Cloning and expression of fragments in E. coli.

Metavinculin differs from vinculin in having an additional insert of 68 to 79 amino acids in length in the C-terminal half of the molecule. Cross-species comparison of metavinculin sequences from pig, man, chicken and frog reveals a division of the insert into two parts: the first variable and the second highly conserved. The longest insert, 79 amino acids, was found in Xenopus laevis. Three different C-terminal constructs of vinculin and metavinculin over-expressed in E. coli could be purified by column chromatography. Two-dimensional gel electrophoresis and peptide analysis revealed pI values between 8.35 and 10.25 for the recombinant proteins. Biochemical and structural features of the metavinculin-specific sequence and the conserved vinculin/metavinculin carboxy-terminus are discussed.

Amino Acid Sequence↗
PubMed1993

Smooth muscle differentiation in human myometrium and uterine leiomyoma.

Smooth muscle differentiation has been analysed in human myometrium and leiomyoma by Western blotting with antibodies to smooth muscle specific proteins. No differences in the expression of h-caldesmon, metavinculin, desmin, alpha-smooth muscle actin and calponin were observed. The technique of two-dimensional gel electrophoresis was used, therefore, to further analyse differences between normal smooth muscle cells and their neoplastic counterparts. By comparing the protein patterns of normal myometrium and leiomyoma, it was possible to identify a protein with a molecular weight of approximately 27 kD that is selectively expressed in normal uterine smooth muscle cells. This protein proved to be a low molecular weight variant of calponin, a smooth muscle specific protein of as yet unknown function. Its immediate downregulation in tissue culture of normal myometrium points to a possible role in the process of dedifferentiation.

Adult↗
PubMed1993

Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle.

The sarcolemma of the smooth muscle cell displays two alternating structural domains in the electron microscope: densely-staining plaques that correspond to the adherens junctions and intervening uncoated regions which are rich in membrane invaginations, or caveolae. The adherens junctions serve as membrane anchorage sites for the actin cytoskeleton and are typically marked by antibodies to vinculin. We show here by immunofluorescence and immunoelectron microscopy that dystrophin is specifically localized in the caveolae-rich domains of the smooth muscle sarcolemma, together with the caveolae-associated molecule caveolin. Additional labeling experiments revealed that beta 1 integrin and fibronectin are confined to the adherens junctions, as indicated by their codistribution with vinculin and tensin. Laminin, on the other hand, is distributed around the entire cell perimeter. The sarcolemma of the smooth muscle cell is thus divided into two distinct domains, featuring different and mutually exclusive components. This simple bipartite domain organization contrasts with the more complex organization of the skeletal muscle sarcolemma: smooth muscle thus offers itself as a useful system for localizing, among other components, potential interacting partners of dystrophin.

Animals↗
PubMed1993

Calponin and SM 22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development.

Calponin and SM 22 are two proteins related in sequence that are particularly abundant in smooth muscle cells. Here, the distribution patterns of calponin and SM 22 were compared with that of other smooth muscle contractile and cytoskeletal components in the avian embryo using immunofluorescence microscopy and immunoblotting. Like myosin-light-chain kinase and heavy caldesmon, both calponin and SM 22 were more or less exclusively found in smooth muscle cells, during embryonic development and in the adult. Labelling of other cell types including striated muscle was not observed. In contrast, tropomyosin, smooth muscle alpha-actin, filamin and desmin could also be detected in many other cell types in addition to smooth muscles, at least during part of embryonic life. Calponin and SM 22 appeared almost synchronously during the differentiation of all smooth muscle cell populations, though with a slight time difference in the case of the aorta. The appearance of calponin, SM 22 and heavy caldesmon was generally delayed in relation to desmin, tropomyosin, smooth muscle alpha-actin, myosin-light-chain kinase and filamin and a marked increase in abundance of these proteins was observed in the late embryo and in the adult. From these observations we can conclude that both calponin and SM 22 belong to a group of late differentiation determinants in smooth muscle and may constitute convenient and reliable markers to follow the differentiation of most, if not all, smooth muscle cell populations.

Actins↗
PubMed1993

Actin and cell movement.

The primary locomotory organelle of non-muscle, eukaryotic cells is the lamellipodium, a thin layer of cytoplasm that exhibits active protrusive activity. Earlier studies have implicated actin polymerization in the formation of lamellipodia, whereby actin monomers insert at the front and dissociate at the rear, in a treadmilling fashion. However, other models based on gel swelling and a breakdown of actin networks at the site of protrusion of the lamellipodium have also been proposed. By employing videomicroscopy and electron microscopy of the same cells, in this case mouse macrophages, it could be shown that lamellipodial protrusion is directly linked with the growth of dense actin meshworks. The gel swelling and cortical breakdown models are not supported by this data. Using rapidly locomoting fish keratocytes, Theriot and Mitchison (Nature 352, 126-131, 1991) recently obtained results that they interpreted as supporting a form of actin filament dynamics different from treadmilling. In their new "nucleation release model" the actin filaments in the lamellipodium are predicted as being very short (less than 0.5 micron) and randomly organized. We have now investigated the ultrastructure of the keratocyte cytoskeleton. Our results show that the actin filaments in these cells are very long and organized in dense and regular, more or less orthogonal networks. A gradient of filament density across the rear part of the lamellipodium suggests that the filaments are graded in length. These data support a treadmilling type model for the eratocyte.

Actins↗